Antibodies that recognize red blood cell antigens

ABSTRACT

Compositions and methods of using antibodies that are able to recognize single amino acid polymorphisms in a protein are provided. Compositions are disclosed which may be used for blood typing or to block hemolytic transfusion reactions and/or hemolytic disease of the fetus and newborn.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to International Application No.PCT/US2015/041416, filed Jul. 21, 2015, which claims the benefit ofpriority to U.S. Provisional Patent Application No. 62/027,207, filedJul. 21, 2014, and to U.S. Provisional Patent Application No.62/120,248, filed Feb. 24, 2015, each of which is incorporated herein byreference in its entirety as if fully set forth herein.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is B212-0002US Sequence Listing_ST25.txt. The textfile is 61 KB, created on Jun. 3, 2018, and is being submittedelectronically via EFS-Web.

FIELD

The invention relates to compositions and methods related to antibodiesthat recognize red blood cell antigens. In particular, the antibodies ofthe invention recognize single amino acid polymorphisms in a protein,such as those that occur on cell surface antigens on red blood cells,for example, the human Kell glycoproteins. The antibodies of the presentinvention can be used for diagnostic or therapeutic uses.

BACKGROUND

Transfusion is a life-saving therapy, given to a large number ofpatients for a wide variety of medical indications. In the United Statesof America alone, approximately 5 million patients (i.e. 1 out of every70 Americans) are transfused with red blood cells (RBCs) each year. Inaddition to the well-known ABO and RhD blood group antigen systems,there are in excess of 300 known RBC antigens that vary from person toperson. Thus, any non-autologous transfusion represents an exposure to amultiplicity of antigenic differences. The immune system of sometransfusion recipients will react to the foreign alloantigens andgenerate alloantibodies. Once a patient has an antibody against an RBCalloantigen, then they are designated “incompatible” with donor RBCsthat express that antigen.

Transfusion of incompatible blood is avoided, because the antibodies candestroy the transfused RBCs. The major problem is not just thatdestroying the RBCs obviates the potential therapeutic effect, but moreimportantly, the process of RBC destruction by recipient antibodies canbe a profound toxic insult to the recipient, leading to myriadpathological outcomes, including: electrolyte disturbance, hemodynamicdysregulation and instability, kidney failure, coagulopathy, and deathin extreme cases. In aggregate, these pathologies are referred to as ahemolytic transfusion reaction (HTR). Avoiding HTRs is the primary goalof blood banks around the world, and represents an entire field ofimmunohematology (e.g. characterizing patient alloantibodies as theyevolve with each transfusion, and providing compatible RBCs notrecognized by a patient's antibodies).

The majority of transfused patients are typically being treated for aninjury or transient illness, from which they subsequently recover, andno longer require transfusion. In such patients, avoiding incompatibletransfusion is an issue of blood bank logistics, and sufficient RBCs canbe provided to such patients by monitoring the antibody response as itevolves and identifying/acquiring units of RBCs lacking the antigens towhich the patients have alloantibodies. However, a subset of patientsrequire chronic transfusion therapy, in some cases for the remainder oftheir lives. For example, genetic abnormalities in RBC production(mostly hemoglobinopatheis) lead to lifelong needs for RBC transfusionsupport (e.g. Sickle cell disease (SCD), alpha and beta thallesemia,Dianond Blackfan anemia, Faconi anemia, etc). As an example, patientswith SCD often have weekly prophylactic transfusions, exposing them to apanoply of different antigens. Up to 50% of SCD patients becomealloimmunized to at least one alloantigen, and once a patient becomesimmunized to one alloantigen, they are more likely to become immunizedto additional antigens. Rates of alloimmunization can be mitigated byprematching to select matched blood group antigens (e.g. Kell, Kidd,Duffy, and others), however such pre-matching is often not feasible andis very costly. In addition, the matching process can delay the deliveryof blood, which may have significant negative consequences if thepatient is being treated for a clinical crisis episode.

The more antigens against which a given patient becomes alloimmunized,the more difficult it becomes to find a sufficient number of compatibleRBC transfusions to meet the patient's clinical needs. In some cases,compatible RBCs do not become available quickly enough to properly carefor the patient, and in extreme cases, alloimmunized patients may diefor wont of sufficient compatible RBCs.

A second disease that can result from patient alloimunization againstRBC antigens is hemolytic disease of the fetus and newborn (HDFN). Inthis case, a pregnant mother has alloantibodies against an antigenexpressed by a fetus she is carrying in her womb. The antibodies cancross the placenta, and destroy fetal RBCs, resulting in fetal anemia,maldevelopment, and in serve cases, death. In HDFN, the mother herselfdoes not become anemic, as the alloantigen in question is not on her ownRBCs, but only on those of the fetus. The frequency of HDFN hasdecreased with the use of anti-D Ig, however alloimmunization stilloccurs against RhD. Moreover, there is no prophylaxis currentlyavailable for antigens such as Kell, Kidd, Duffy etc. Once a woman isalloimmunized and pregnant with an antigen positive fetus, the primarytreatment is intrauterine transfusions with RBC negative blood andsymptomatic treatment.

The inability of current technologies to provide sufficient units ofcompatible RBCs for alloimmunized patients, resulting in morbidity andmortality due to lack of compatible blood, is a primary medical needaddressed by the current disclosure. A secondary application of thepresent disclosure is for the treatment of pregnant women whose fetusesare suffering HDFN.

SUMMARY

Described herein are compositions and methods related to monoclonalantibodies capable of distinguishing single amino acid determinants onan antigen, in particular, antigens found on the surfaces of red bloodcells. Such antibodies can be used for diagnostic applications such asRBC typing. In other embodiments, the compositions and methods disclosedherein can be used therapeutically, such as to block hemolytictransfusion reactions.

In a first aspect, disclosed herein is an isolated antibody or fragmentthereof that binds to a red blood cell surface antigen and blocks ahemolytic transfusion reaction.

In various embodiments of this aspect, the antibody recognizes theepitope created by a single amino acid polymorphism.

In other embodiments of this aspect, the epitope/antigen is a member ofthe Kell blood group antigen system, for example, KEL1, KEL2, KEL3,KEL4, KEL5, KEL6, or KEL7. In particular embodiments, the Kell bloodgroup antigen is K, Kp^(b), or Js^(b).

In further embodiments of this aspect, the antibody or fragment thereofcomprises a heavy chain comprising at least one CDR selected from thegroup of CDR sequences shown in FIG. 1.

In yet further embodiments of this aspect, the antibody or fragmentthereof comprises a light chain comprising at least one CDR selectedfrom the group of CDR sequences shown in FIG. 2.

In other embodiments of this aspect, the antibody or fragment thereofcomprises a heavy chain comprising one, two, or three CDR(s) selectedfrom the group of CDR sequences shown in FIG. 1.

In other embodiments of this aspect, the antibody or fragment thereofcomprises a light chain comprising one, two, or three CDR(s) selectedfrom the group of CDR sequences shown in FIG. 2.

In other embodiments of this aspect, the antibody or fragment thereofcomprises a heavy chain comprising at least a portion of the sequenceshown in FIG. 1.

In other embodiments of this aspect, the antibody or fragment thereofcomprises a light chain comprising at least a portion of the sequenceshown in FIG. 2.

In other embodiments of this aspect, the antibody or fragment thereof isselected from the group consisting of: (a) a whole immunoglobulinmolecule; (b) an scFv; (c) a Fab fragment; (d) an F(ab′)2; and (e) adisulfide linked Fv.

In various of the above aspects and embodiments, the antibody orfragment comprises a heavy chain immunoglobulin constant domain selectedfrom the group consisting of: (a) a human IgM constant domain; (b) ahuman IgG1 constant domain; (c) a human IgG2 constant domain; (d) ahuman IgG3 constant domain; (e) a human IgG4 constant domain; and (f) ahuman IgA1/2 constant domain.

In various of the above aspects and embodiments, the antibody orfragment thereof comprises a light chain immunoglobulin constant domainselected from the group consisting of: (a) a human Ig kappa constantdomain; and (b) a human Ig lambda constant domain.

In various of the above aspects and embodiments, the antibody orfragment thereof is a mouse IgG1, IgG2a, IgG2b, IgG2c, or IgG3.

In various of the above aspects and embodiments, the antibody orfragment thereof comprises mutations in the constant region. Examples ofsuch mutations include, but are not limited to, mutations in theconstant region that alter binding to Fc Receptors, alter fixation ofcomplement, or alter the ability to cross the placenta into fetalcirculation.

In various of the above aspects and embodiments, the antibody orfragment thereof comprises alterations in glycosyslation of the antibodyor fragment thereof. Examples of such alterations in glycosyslationinclude, but are not limited to, alterations in fucosylation,sialylation, or modification of GlcNAC, glucose, or galactose.

In various of the above aspects and embodiments, the antibody orfragment thereof binds to an antigen with an affinity constant (K_(D))of less than 1×10⁻⁸ M.

In various of the above aspects and embodiments, the antibody orfragment thereof binds to an antigen with an affinity constant (K_(D))of less than 1×10⁻⁹ M.

In a second aspect, disclosed herein is a method of preventing orreducing a hemolytic transfusion reaction by administering atherapeutically effective amount of the antibody or fragment thereofdisclosed above to a subject in need thereof prior to a transfusion withred blood cells. In some embodiments, the antibody or fragment isadministered to the subject prior to the transfusion. In otherembodiments, the red blood cells for transfusion are treated with theantibody or fragment thereof prior to transfusion into the subject. Insome embodiments, the antibody or fragment thereof is administeredintravenously (IV), subcutaneously (SC), or intramuscularly (IM). Insome embodiments, the antibody or fragment thereof is administered in anamount in the range of 1 to 100 milligrams per kilogram of the subject'sbody weight.

In a related aspect, disclosed herein is a method of treating,preventing, or reducing a hemolytic transfusion reaction, the methodcomprising the steps of administering a therapeutically effective amountof an antibody or fragment thereof to a subject in need thereof prior toa transfusion with donor red blood cells, wherein said antibody orfragment thereof binds to a red blood cell antigen on the donor redblood cells to block the binding of a hemolytic antibody and whereinsaid antibody or fragment thereof does not itself cause destruction ofdonor red blood cells, thereby treating, preventing, or reducing ahemolytic transfusion reaction. In some embodiments, the antibody orfragment is administered to the subject prior to the transfusion. Inother embodiments, the red blood cells for transfusion are treated withthe antibody or fragment thereof prior to transfusion into the subject.In various embodiments of this aspect, the antibodies or fragmentsthereof disclosed above are used. In some embodiments, the antibody orfragment thereof is administered intravenously (IV), subcutaneously(SC), or intramuscularly (IM). In some embodiments, the antibody orfragment thereof is administered in an amount in the range of 1 to 100milligrams per kilogram of the subject's body weight.

In a third aspect, disclosed herein is an expression vector comprising anucleic acid encoding the antibody or fragment thereof disclosed above.

In some embodiments of this aspect, the expression vector is in a hostcell, which can include a bacterial cell or a eukaryotic cell, such as amammalian cell.

In a fourth aspect, disclosed herein is an antibody or fragment thereofcomprises a heavy chain comprising at least one CDR selected from thegroup of CDR sequences shown in FIGS. 1, 3, and 5.

In a fifth aspect, disclosed herein is an antibody or fragment thereofcomprises a light chain comprising at least one CDR selected from thegroup of CDR sequences shown in FIGS. 2, 4, and 6.

In a sixth aspect, disclosed herein is an antibody or fragment thereofcomprises a heavy chain comprising one, two, or three CDR(s) selectedfrom the group of CDR sequences shown in FIGS. 1, 3, and 5.

In a seventh aspect, disclosed herein is an antibody or fragment thereofcomprises a light chain comprising one, two, or three CDR(s) selectedfrom the group of CDR sequences shown in FIGS. 2, 4, and 6.

In an eighth aspect, disclosed herein is an antibody or fragment thereofcomprises a heavy chain comprising the sequence shown in FIG. 1, FIG. 3,or FIG. 5.

In a ninth aspect, disclosed herein is an antibody or fragment thereofcomprises a light chain comprising the sequence shown in FIG. 2, FIG. 4,or FIG. 6.

In a tenth aspect, disclosed herein is an antibody or fragment thereofcomprises a heavy chain comprising the sequence of FIG. 1 and a lightchain sequence of FIG. 2.

In a eleventh aspect, disclosed herein is an antibody or fragmentthereof comprises a heavy chain comprising the sequence of FIG. 3 and alight chain sequence of FIG. 4.

In a twelfth aspect, disclosed herein is an antibody or fragment thereofcomprises a heavy chain comprising the sequence of FIG. 5 and a lightchain sequence of FIG. 6.

In some embodiments of these aspects, the antibody or fragment thereofis selected from the group consisting of: (a) a whole immunoglobulinmolecule; (b) an scFv; (c) a Fab fragment; (d) an F(ab′)2; and (e) adisulfide linked Fv.

In various of the above aspect and embodiments, the antibody or fragmentcomprises a heavy chain immunoglobulin constant domain selected from thegroup consisting of: (a) a human IgM constant domain; (b) a human IgG1constant domain; (c) a human IgG2 constant domain; (d) a human IgG3constant domain; (e) a human IgG4 constant domain; and (f) a humanIgA1/2 constant domain.

In various of the above aspect and embodiments, the antibody or fragmentthereof comprises a light chain immunoglobulin constant domain selectedfrom the group consisting of: (a) a human Ig kappa constant domain; and(b) a human Ig lambda constant domain.

In various of the above aspect and embodiments, the antibody or fragmentthereof is a mouse IgG1, IgG2a, IgG2b, IgG2c, or IgG3.

In various of the above aspect and embodiments, the antibody or fragmentthereof comprises mutations in the constant region. Examples of suchmutations include, but are not limited to, mutations in the constantregion that alter binding to Fc Receptors, alter fixation of compliment,or alter the ability to cross the placenta into fetal circulation.

In various of the above aspect and embodiments, the antibody or fragmentthereof comprises alterations in glycosyslation of the antibody orfragment thereof. Examples of such alterations in glycosyslationinclude, but are not limited to, alterations in fucosylation,sialylation, or modification of GlcNAC, glucose, or galactose.

In various of the above aspect and embodiments, the antibody or fragmentthereof binds to an antigen with an affinity constant (K_(D)) of lessthan 1×10⁻⁸ M.

In various of the above aspect and embodiments, the antibody or fragmentthereof binds to an antigen with an affinity constant (K_(D)) of lessthan 1×10⁻⁹ M.

In a thirteenth aspect, disclosed herein is a method of generating anantibody or fragment thereof that binds to a red blood cell surfaceantigen or fragment thereof, the method comprising the steps of: (a)generating mice expressing a human red blood cell surface antigen on itsred blood cells; (b) immunizing wild type mice by transfusing red bloodcells from the mice expressing the human red blood cell surface antigenon its red blood cells; and (c) using splenocytes from the immunizedmice to generate monoclonal antibodies. In some embodiments of thisaspect, the epitope/antigen is a member of the Kell blood group antigensystem, for example, KEL1, KEL2, KEL3, KEL4, KEL5, KEL6, or KEL7. Inparticular embodiments, the Kell blood group antigen is KEL1 (K) or KEL4(Kp^(b)).

In a fourteenth aspect, disclosed herein is an expression vectorcomprising any one of the nucleic acids shown in FIGS. 1-6.

In some embodiments of this aspect, the expression vector is in a hostcell, which can include a bacterial cell or a eukaryotic cell, such as amammalian cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the heavy chain sequence (SEQ ID NOs: 1, 2, and 3) ofmonoclonal antibodies Puma 1 and 2 directed to KEL1 (K). The shadingindicates where the highly variable region begins. The CDR regions(CDR1: DYYMK, SEQ ID NO: 25; CDR2: DLNPNNGDTFYNQKFKG, SEQ ID NO: 26; andCDR3: CAREAGSSFGSSCNYWG, SEQ ID NO: 27) of the heavy chain areunderlined.

FIG. 2 shows the light chain sequence (SEQ ID NOs: 5, 6, and 7) ofmonoclonal antibodies Puma 1 and 2 directed to KEL1 (K). The shadingindicates where the highly variable region begins. The CDR regions(CDR1: KASQTVSKDVA, SEQ ID NO: 28; CDR2: YASNRYT, SEQ ID NO: 29; andCDR3: QQDYSS, SEQ ID NO: 30) of the light chain are underlined.

FIG. 3 shows the heavy chain sequence (SEQ ID NOs: 8, 9, and 10) ofmonoclonal antibody Puma 3 directed to a common Kell epitope. Theshading indicates where the highly variable region begins. The CDRregions (CDR1: SYGVY, SEQ ID NO: 31; CDR2: IIWGDGSTNYQSVLRS, SEQ ID NO:32; and CDR3: RGDYDVA, SEQ ID NO: 33) of the heavy chain are underlined.

FIG. 4 shows the light chain sequence (SEQ ID NOs: 12, 13, and 14) ofmonoclonal antibody Puma 3 directed to a common Kell epitope. Theshading indicates where the highly variable region begins. The CDRregions (CDR1: KASQTVSEVGTSLMH, SEQ ID NO: 34; CDR2: RTSNLEA, SEQ ID NO:35; and CDR3: QQS, SEQ ID NO: 36) of the light chain are underlined.

FIG. 5 shows the heavy chain sequence (SEQ ID NOs: 15, 16, and 17) ofmonoclonal antibody Puma 4 directed to KEL4 (Kp^(b)). The shadingindicates where the highly variable region begins. The CDR regions(CDR1: NYWMN, SEQ ID NO: 37; CDR2: EIRLNSNNYATHYAESVKG, SEQ ID NO: 38;and CDR3: NWDFAW, SEQ ID NO: 39) of the heavy chain are underlined.

FIG. 6 shows the light chain sequence (SEQ ID NOs: 18, 19, and 20) ofmonoclonal antibody Puma 4 directed to KEL4 (Kp^(b)). The CDR regions(CDR1: KASQDVSTVVA, SEQ ID NO: 40; CDR2: WASTRHT, SEQ ID NO: 41; andCDR3: QQHYT, SEQ ID NO: 42) of the light chain are underlined.

FIGS. 7A, 7B shows the specificity of monoclonal antibody Puma 1.

FIGS. 8A, 8B shows the specificity of monoclonal antibody Puma 2.

FIGS. 9A-9C shows the specificity of monoclonal antibody Puma 3.

FIGS. 10A-10I shows the specificity of monoclonal antibody Puma 4.

FIGS. 11A-11B shows an overview of protective antibody (protectabody)therapy.

FIG. 12 shows potential modifications and features of protectiveantibody (protectabody) structure.

FIG. 13 shows that PUMA 1 has the capacity to cause a hemolytictransfusion reaction (HTR), in a mouse model, in vivo.

FIG. 14 shows the differential ability of different IgG subclassversions of PUMA 1 to induce a hemolytic transfusion reaction (HTR).

FIG. 15 shows the effect of PUMA 1 IgG3 on blocking hemolysis caused byPUMA 1 IgG2a; these data demonstrate in vivo efficacy, showing theblocking of a HTR by a less hemolytic engineered form.

FIGS. 16A, 16B show the sequences of humanization of PUMA1 to humanIgG1, IgG2, IgG3, and IgG4 (FIG. 16A, SEQ ID NOs: 21-24) and thealignment of these sequences (FIG. 16B).

FIG. 17 shows recombinant generation of a humanized form of PUMA1 andits ability to bind to antigen positive RBCs, demonstrating amaintenance of binding after humanization of the IgG constant region.

DETAILED DESCRIPTION

In one embodiment, the present disclosure describes the isolation ofantibodies with sufficient fine specificity to recognize conformationalchanges brought about by single amino acid polymorphisms in a givenprotein. Such antibodies are difficult to isolate for a number ofreasons. First, the antigens are typically transmembrane proteins withconformational requirements of being expressed on the cell surface,precluding isolation of large amounts of cell free antigen forimmunization. Second, expression in cell lines typically results in thedesired antigen, but also a great number of additional foreign antigens,which can dominate the immune response and limit antibodies of thedesired specificity. The present invention provides methods andcompostions that circumvent many of these limitations to provide new RBCtyping reagents.

One principle behind the therapeutic application of the technologydisclosed herein is the engineering of molecules that mask offendingantigens on RBCs and thereby block recipient/maternal alloantibodiesfrom binding to and destroying the RBCs. In the normal clinicalsituation of incompatible RBCs described above, an HTR occurs whenrecipient antibodies bind to donor RBC antigens, resulting in RBCdestruction (FIG. 11A). The general concept is to engineer recombinantantibodies that bind to the antigen(s) in question but are modified suchthat their Fc region no longer binds to Fc receptors or complement(green bars, FIGS. 11A, 11B), thus rendering it “non-hemolytic” (FIG.11B). Such an engineered antibody will bind to the same antigenrecognized by hemolytic antibodies in the recipient and thus block theantigen with a non-hemolytic entity. In this way, the engineeredantibody will prevent hemolysis of the RBCs and allow transfusiondespite alloantibodies in the recipient, which would otherwise behemolytic. The modified protective antibody (called protectabodieshereafter) will retain an Fc region to provide long circulatoryhalf-life; however, modifications may include (but not be limited to),removing Fc Receptor binding of activating receptors, increasing bindingto FcgRIIb (an inhibitory Fc receptor), removing complement binding, andadding complement inhibitory domains (FIG. 12).

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only,and is not intended to be limiting. As used in this specification andthe appended claims, the singular forms “a”, “an” and “the” includeplural references unless the content clearly dictates otherwise.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein.

As used herein, the term “hemolytic transfusion reaction” refersgenerally to a complication that can occur after a transfusion of bloodin which the red blood cells that were given in the transfusion aredestroyed by the patient's immune system (antibodies to the donor RBCs).Symptoms of a hemolytic transfusion reaction may include: back pain,bloody urine, chills, fainting or dizziness, fever, flank pain, andflushing of the skin. In severe cases, hemolytic transfusion reactionscan lead to organ failure, coagulation defects, and/or death.

As used herein, a “hemolytic antibody” is an antibody, typically arecipient or maternal alloantibody, that binds to a red blood cellantigen on donor red blood cells from a transfusion and causingdestruction or hemolysis of the transfused donor red blood cells.

A “protective antibody” or “protectabody” refers generally to anantibody that will bind to an antigen recognized by a hemolytic antibodyin a recipient, but will not cause red blood cell destruction orhemolysis. In this way, the protective antibody blocks the hemolyticantigen with a non-hemolytic entity. Generally, a protective antibody ismodified to render it non-hemolytic as described herein. Suchmodifications may include: removing Fc Receptor binding of activatingreceptors, increasing binding to FcgRIIb (an inhibitory Fc receptor),removing complement binding, and adding complement inhibitory domains.

“Subject,” “mammalian subject,” or “patient” are used interchangeablyand refer to mammals such as human patients and non-human primates, aswell as experimental animals such as rabbits, rats, and mice, cows,horses, goats, and other animals. Animals include all vertebrates, e.g.,mammals and non-mammals, such as mice, sheep, dogs, cows, avian species,ducks, geese, pigs, chickens, amphibians, and reptiles.

“Treating” or “treatment” refers generally to either (i) the prevention,e.g., prophylaxis, or (ii) the reduction or elimination of symptoms of adisease of interest, e.g., therapy. Treating a subject with thecompositions of the invention can prevent or reduce the risk of thesubject suffering from a hemolytic transfusion reaction (HTR). Treatmentcan be prophylactic (to prevent or delay the onset of the disease, or toprevent the manifestation of clinical or subclinical symptoms thereof)or therapeutic suppression or alleviation of symptoms after themanifestation of the disease.

“Preventing” or “prevention” refers to prophylactic administration withcompositions of the invention.

“Therapeutically-effective amount” or “an amount effective to reduce theeffects of a disease” or “an effective amount” refers to an amount of anantibody composition that is sufficient to prevent a hemolytictransfusion reaction or to alleviate (e.g., mitigate, decrease, reduce)at least one of the symptoms associated with this condition. It is notnecessary that the administration of the composition eliminate thesymptoms of a hemolytic transfusion reaction, as long as the benefits ofadministration of the composition outweigh the detriments. Likewise, theterms “treat” and “treating” in reference to a hemolytic transfusionreaction, as used herein, are not intended to mean that the subject isnecessarily cured of the condition or that all clinical signs thereofare eliminated, only that some alleviation or improvement in thecondition of the subject is effected by administration of thecomposition.

Polypeptides

The term “polypeptide” or “peptide” refers to a polymer of amino acidswithout regard to the length of the polymer; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide. This term also does not specify or exclude post-expressionmodifications of polypeptides, for example, polypeptides which includethe covalent attachment of glycosyl groups, acetyl groups, phosphategroups, lipid groups and the like are expressly encompassed by the termpolypeptide. Also included within the definition are polypeptides whichcontain one or more analogs of an amino acid (including, for example,non-naturally occurring amino acids, amino acids which only occurnaturally in an unrelated biological system, modified amino acids frommammalian systems etc.), polypeptides with substituted linkages, as wellas other modifications known in the art, both naturally occurring andnon-naturally occurring.

The term “isolated protein,” “isolated polypeptide,” or “isolatedpeptide” is a protein, polypeptide or peptide that by virtue of itsorigin or source of derivation (1) is not associated with naturallyassociated components that accompany it in its native state, (2) is freeof other proteins from the same species, (3) is expressed by a cell froma different species, or (4) does not occur in nature. Thus, a peptidethat is chemically synthesized or synthesized in a cellular systemdifferent from the cell from which it naturally originates will be“isolated” from its naturally associated components. A protein may alsobe rendered substantially free of naturally associated components byisolation, using protein purification techniques well known in the art.

The terms “polypeptide”, “protein”, “peptide,” “antigen,” or “antibody”within the meaning of the present invention, includes variants, analogs,orthologs, homologs and derivatives, and fragments thereof that exhibita biological activity, generally in the context of being able to inducean immune response in a subject, or bind an antigen in the case of anantibody.

The polypeptides of the invention include an amino acid sequence derivedfrom Kell system antigens or fragments thereof, corresponding to theamino acid sequence of a naturally occurring protein or corresponding tovariant protein, i.e., the amino acid sequence of the naturallyoccurring protein in which a small number of amino acids have beensubstituted, added, or deleted but which retains essentially the sameimmunological properties. In addition, such derived portion can befurther modified by amino acids, especially at the N- and C-terminalends to allow the polypeptide or fragment to be conformationallyconstrained and/or to allow coupling to an immunogenic carrier afterappropriate chemistry has been carried out. The polypeptides of thepresent invention encompass functionally active variant polypeptidesderived from the amino acid sequence of Kell system antigens in whichamino acids have been deleted, inserted, or substituted withoutessentially detracting from the immunological properties thereof, i.e.such functionally active variant polypeptides retain a substantialpeptide biological activity.

In one embodiment, such functionally active variant polypeptides exhibitat least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity toan amino acid sequence of the blood group antigens disclosed herein.Sequence similarity for polypeptides, which is also referred to assequence identity, is typically measured using sequence analysissoftware. Protein analysis software matches similar sequences usingmeasures of similarity assigned to various substitutions, deletions andother modifications, including conservative amino acid substitutions.For instance, GCG contains programs such as “Gap” and “Bestfit” whichcan be used with default parameters to determine sequence homology orsequence identity between closely related polypeptides, such ashomologous polypeptides from different species of organisms or between awild type protein and a mutein thereof. See, e.g., GCG Version 6.1.Polypeptide sequences also can be compared using FASTA using default orrecommended parameters, a program in GCG Version 6.1. FASTA (e.g.,FASTA2 and FASTA3) provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.132:185-219 (2000)). An alternative algorithm when comparing a sequenceof the invention to a database containing a large number of sequencesfrom different organisms is the computer program BLAST, especiallyblastp or tblastn, using default parameters. See, e.g., Altschul et al.,J. Mol. Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res.25:3389-402 (1997).

Functionally active variants comprise naturally occurring functionallyactive variants such as allelic variants and species variants andnon-naturally occurring functionally active variants that can beproduced by, for example, mutagenesis techniques or by direct synthesis.

A functionally active variant can exhibit, for example, at least 60%,65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acidsequence of a Kell system or other antigen disclosed herein, and yetretain a biological activity. Where this comparison requires alignment,the sequences are aligned for maximum homology. The site of variationcan occur anywhere in the sequence, as long as the biological activityis substantially similar to the Kell system or other antigens disclosedherein, e.g., ability to induce a tolerance reponse. Guidance concerninghow to make phenotypically silent amino acid substitutions is providedin Bowie et al., Science, 247: 1306-1310 (1990), which teaches thatthere are two main strategies for studying the tolerance of an aminoacid sequence to change. The first strategy exploits the tolerance ofamino acid substitutions by natural selection during the process ofevolution. By comparing amino acid sequences in different species, theamino acid positions which have been conserved between species can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions in which substitutionshave been tolerated by natural selection indicate positions which arenot critical for protein function. Thus, positions tolerating amino acidsubstitution can be modified while still maintaining specificimmunogenic activity of the modified polypeptide.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site-directed mutagenesis oralanine-scanning mutagenesis can be used (Cunningham et al., Science,244: 1081-1085 (1989)). The resulting variant polypeptides can then betested for specific biological activity.

According to Bowie et al., these two strategies have revealed thatproteins are surprisingly tolerant of amino acid substitutions. Theauthors further indicate which amino acid changes are likely to bepermissive at certain amino acid positions in the protein. For example,the most buried or interior (within the tertiary structure of theprotein) amino acid residues require nonpolar side chains, whereas fewfeatures of surface or exterior side chains are generally conserved.

Methods of introducing a mutation into amino acids of a protein is wellknown to those skilled in the art. (See, e.g., Ausubel (ed.), CurrentProtocols in Molecular Biology, John Wiley and Sons, Inc. (1994); T.Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989)).

Mutations can also be introduced using commercially available kits suchas “QuikChange Site-Directed Mutagenesis Kit” (Stratagene) or directlyby peptide synthesis. The generation of a functionally active variant toan peptide by replacing an amino acid which does not significantlyinfluence the function of said peptide can be accomplished by oneskilled in the art.

A type of amino acid substitution that may be made in the polypeptidesof the invention is a conservative amino acid substitution. A“conservative amino acid substitution” is one in which an amino acidresidue is substituted by another amino acid residue having a side chainR group) with similar chemical properties (e.g., charge orhydrophobicity). In general, a conservative amino acid substitution willnot substantially change the functional properties of a protein. Incases where two or more amino acid sequences differ from each other byconservative substitutions, the percent sequence identity or degree ofsimilarity may be adjusted upwards to correct for the conservativenature of the substitution. Means for making this adjustment arewell-known to those of skill in the art. See e.g. Pearson, Methods Mol.Biol. 243:307-31 (1994).

Examples of groups of amino acids that have side chains with similarchemical properties include 1) aliphatic side chains: glycine, alanine,valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains:serine and threonine; 3) amide-containing side chains: asparagine andglutamine; 4) aromatic side chains: phenylalanine, tyrosine, andtryptophan; 5) basic side chains: lysine, arginine, and histidine; 6)acidic side chains: aspartic acid and glutamic acid; and 7)sulfur-containing side chains: cysteine and methionine. Preferredconservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.

Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al., Science 256:1443-45 (1992). A “moderately conservative”replacement is any change having a nonnegative value in the PAM250log-likelihood matrix.

A functionally active variant can also be isolated using a hybridizationtechnique. Briefly, DNA having a high homology to the whole or part of anucleic acid sequence encoding the peptide, polypeptide or protein ofinterest, e.g. Kell system antigens, is used to prepare a functionallyactive peptide. Therefore, a polypeptide of the invention also includesentities which are functionally equivalent and which are encoded by anucleic acid molecule which hybridizes with a nucleic acid encoding anyone of the Kell system antigens or a complement thereof. One of skill inthe art can easily determine nucleic acid sequences that encode peptidesof the invention using readily available codon tables. As such, thesenucleic acid sequences are not presented herein.

Nucleic acid molecules encoding a functionally active variant can alsobe isolated by a gene amplification method such as PCR using a portionof a nucleic acid molecule DNA encoding a peptide, polypeptide, protein,antigen, or antibody of interest, e.g. Kell system antigens, as theprobe.

For the purpose of the present invention, it should be considered thatseveral polypeptides or antigens of the invention may be used incombination. All types of possible combinations can be envisioned. Thesame sequence can be used in several copies on the same polypeptidemolecule, or wherein peptides of different amino acid sequences are usedon the same polypeptide molecule; the different peptides or copies canbe directly fused to each other or spaced by appropriate linkers. Asused herein the term “multimerized (poly)peptide” refers to both typesof combination wherein polypeptides of either different or the sameamino acid sequence are present on a single polypeptide molecule. From 2to about 20 identical and/or different peptides can be thus present on asingle multimerized polypeptide molecule.

In one embodiment of the invention, a peptide, polypeptide, protein, orantigen of the invention is derived from a natural source and isolatedfrom a bacterial source. A peptide, polypeptide, protein, or antigen ofthe invention can thus be isolated from sources using standard proteinpurification techniques.

Alternatively, peptides, polypeptides and proteins of the invention canbe synthesized chemically or produced using recombinant DNA techniques.For example, a peptide, polypeptide, or protein of the invention can besynthesized by solid phase procedures well known in the art. Suitablesyntheses may be performed by utilising “T-boc” or “F-moc” procedures.Cyclic peptides can be synthesised by the solid phase procedureemploying the well-known “F-moc” procedure and polyamide resin in thefully automated apparatus. Alternatively, those skilled in the art willknow the necessary laboratory procedures to perform the processmanually. Techniques and procedures for solid phase synthesis aredescribed in ‘Solid Phase Peptide Synthesis: A Practical Approach’ by E.Atherton and R. C. Sheppard, published by IRL at Oxford University Press(1989) and ‘Methods in Molecular Biology, Vol. 35: Peptide SynthesisProtocols (ed. M. W. Pennington and B. M. Dunn), chapter 7, pp 91-171 byD. Andreau et al.

Alternatively, a polynucleotide encoding a peptide, polypeptide orprotein of the invention can be introduced into an expression vectorthat can be expressed in a suitable expression system using techniqueswell known in the art, followed by isolation or purification of theexpressed peptide, polypeptide, or protein of interest. A variety ofbacterial, yeast, plant, mammalian, and insect expression systems areavailable in the art and any such expression system can be used.Optionally, a polynucleotide encoding a peptide, polypeptide or proteinof the invention can be translated in a cell-free translation system.

Nucleic acid sequences corresponding to Kell system antigens can also beused to design oligonucleotide probes and used to screen genomic or cDNAlibraries for genes encoding other variants or from other species. Thebasic strategies for preparing oligonucleotide probes and DNA libraries,as well as their screening by nucleic acid hybridization, are well knownto those of ordinary skill in the art. See, e.g., DNA Cloning: Vol. I,supra; Nucleic Acid Hybridization, supra; Oligonucleotide Synthesis,supra; Sambrook et al., supra. Once a clone from the screened libraryhas been identified by positive hybridization, it can be confirmed byrestriction enzyme analysis and DNA sequencing that the particularlibrary insert contains a Kell system antigen gene, or a homologthereof. The genes can then be further isolated using standardtechniques and, if desired, PCR approaches or restriction enzymesemployed to delete portions of the full-length sequence.

Alternatively, DNA sequences encoding the proteins of interest can beprepared synthetically rather than cloned. The DNA sequences can bedesigned with the appropriate codons for the particular amino acidsequence. In general, one will select preferred codons for the intendedhost if the sequence will be used for expression. The complete sequenceis assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence. See, e.g., Edge(1981) Nature 292: 756; Nambair et al. (1984) Science 223: 1299; Jay etal. (1984) J. Biol. Chem. 259: 6311.

Once coding sequences for the desired proteins have been prepared orisolated, they can be cloned into any suitable vector or replicon.Numerous cloning vectors are known to those of skill in the art, and theselection of an appropriate cloning vector is a matter of choice.Examples of recombinant DNA vectors for cloning and host cells whichthey can transform include the bacteriophage λ (E. coli), pBR322 (E.coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus(mammalian cells). See, Sambrook et al., supra; DNA Cloning, supra; B.Perbal, supra. The gene can be placed under the control of a promoter,ribosome binding site (for bacterial expression) and, optionally, anoperator (collectively referred to herein as “control” elements), sothat the DNA sequence encoding the desired protein is transcribed intoRNA in the host cell transformed by a vector containing this expressionconstruction. The coding sequence can or can not contain a signalpeptide or leader sequence. Leader sequences can be removed by the hostin post-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739;4,425,437; 4,338,397. Examples of vectors include pET32a(+) andpcDNA3002Neo.

Other regulatory sequences can also be desirable which allow forregulation of expression of the protein sequences relative to the growthof the host cell. Regulatory sequences are known to those of skill inthe art, and examples include those which cause the expression of a geneto be turned on or off in response to a chemical or physical stimulus,including the presence of a regulatory compound. Other types ofregulatory elements can also be present in the vector, for example,enhancer sequences.

The control sequences and other regulatory sequences can be ligated tothe coding sequence prior to insertion into a vector, such as thecloning vectors described above. Alternatively, the coding sequence canbe cloned directly into an expression vector which already contains thecontrol sequences and an appropriate restriction site.

In some cases it can be necessary to modify the coding sequence so thatit can be attached to the control sequences with the appropriateorientation; i.e., to maintain the proper reading frame. It can also bedesirable to produce mutants or analogs of the protein. Mutants oranalogs can be prepared by the deletion of a portion of the sequenceencoding the protein, by insertion of a sequence, and/or by substitutionof one or more nucleotides within the sequence. Techniques for modifyingnucleotide sequences, such as site-directed mutagenesis, are describedin, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic AcidHybridization, supra.

The expression vector is then used to transform an appropriate hostcell. A number of mammalian cell lines are known in the art and includeimmortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),Madin-Darby bovine kidney (“MDBK”) cells, HEK293F cells, NSO-1 cells, aswell as others. Similarly, bacterial hosts such as E. coli, Bacillussubtilis, and Streptococcus spp., will find use with the presentexpression constructs. Yeast hosts useful in the present inventioninclude, but are not limited to, Saccharomyces cerevisiae, Candidaalbicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis,Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris,Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for usewith baculovirus expression vectors include, but are not limited to,Aedes aegypti, Autographa californica, Bombyx mori, Drosophilamelanogaster, Spodoptera frugiperda, and Trichoplusia ni.

Depending on the expression system and host selected, the proteins ofthe present invention are produced by culturing host cells transformedby an expression vector described above under conditions whereby theprotein of interest is expressed. The protein is then isolated from thehost cells and purified. The selection of the appropriate growthconditions and recovery methods are within the skill of the art.

Kell system antigen protein sequences can also be produced by chemicalsynthesis such as solid phase peptide synthesis, using known amino acidsequences or amino acid sequences derived from the DNA sequence of thegenes of interest. Such methods are known to those skilled in the art.See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis,2nd Ed., Pierce Chemical Co., Rockford, Ill. (1984) and G. Barany and R.B. Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E.Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp.3-254, for solid phase peptide synthesis techniques; and M. Bodansky,Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984) and E.Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis,Biology, supra, Vol. 1, for classical solution synthesis. Chemicalsynthesis of peptides can be preferable if a small fragment of theantigen in question is capable of raising an immunological response inthe subject of interest.

Polypeptides of the invention can also comprise those that arise as aresult of the existence of multiple genes, alternative transcriptionevents, alternative RNA splicing events, and alternative translationaland postranslational events. A polypeptide can be expressed in systems,e.g. cultured cells, which result in substantially the samepostranslational modifications present as when the peptide is expressedin a native cell, or in systems that result in the alteration oromission of postranslational modifications, e.g. glycosylation orcleavage, present when expressed in a native cell.

A peptide, polypeptide, protein, or antigen of the invention can beproduced as a fusion protein that contains other distinct amino acidsequences that are not part of the Kell system antigen sequencesdisclosed herein, such as amino acid linkers or signal sequences orimmunogenic carriers, as well as ligands useful in protein purification,such as glutathione-S-transferase, histidine tag, and staphylococcalprotein A. More than one polypeptide of the invention can be present ina fusion protein. The heterologous polypeptide can be fused, forexample, to the N-terminus or C-terminus of the peptide, polypeptide orprotein of the invention. A peptide, polypeptide, protein, or antigen ofthe invention can also be produced as fusion proteins comprisinghomologous amino acid sequences.

Blood Group Antigen Proteins

Any of a variety of cell surface proteins found on red blood cells maybe used in the practice of the present invention. In one embodiment, theproteins are blood group antigens, such as the Kell system antigens.Information on such antigens and, in particular, soluble forms areavailable in the art, for example, in Ridgwell et al., TransfusionMedicine, 17: 384-394 (2007).

Kell (CD238) is a clinically important human blood group antigen systemcomprising 28 antigens (Daniels et al., 2007, International Society ofBlood Transfusion Committee on Terminology for Red Cell SurfaceAntigens: Cape Town report. Vox Sanguinis, 92, 250-253). The Kellantigens are carried by a single pass type II (cytoplasmic N-terminus)red blood cell membrane glycoprotein. The Kell glycoprotein is expressedin red cells and haematopoietic tissue (bone marrow and foetal liver)and to a lesser extent in other tissues, including brain, lymphoidorgans, heart and skeletal muscle (Russo et al., 2000, Blood, 96,340-346). The K/k (KEL1/KEL2) blood group antigen polymorphism isdetermined by a single nucleotide polymorphism (SNP) resulting in thepresence of methionine (M) or threonine (T), respectively, at amino acid193 of the extracellular C-terminal domain (Lee, 1997, Vox Sanguinis,73, 1-11). The other mostclinically significant antithetical antigensKp^(a)/Kp^(b) (KEL3/KEL4) and Js^(a)/Js^(b) (KEL6/KEL7) are also theresult of SNPs resulting in single amino acid changes in theextracellular domain (Lee, 1997, Vox Sanguinis, 73, 1-11).

Kell system antibodies are known to cause haemolytic transfusionreactions and haemolytic disease of the fetus and newborn (HDFN).Kell-related HDFN may be because of suppression of fetal erythropoiesisin addition to immune destruction of red blood cells as in most othercases of HDFN (Vaughan et al., 1998, New England Journal of Medicine,338, 798-803; Daniels et al., 2003, Transfusion, 43, 115-116). Anti-K(KEL1) is the most commonly encountered immune red cell antibody outsidethe ABO and Rh systems, and other antigens of the Kell blood groupsystem, e.g. k (KEL2), Kp^(a) (KEL3), Kp^(b) (KEL4), Js^(a) (KEL6) andJs^(b) (KEL7) are also capable of stimulating the production ofhaemolytic antibodies and causing HDFN (Daniels, 2002, Human BloodGroups (2nd edn). Blackwell, Oxford).

The Duffy (Fy, CD234) blood group antigens are carried by a type IIImembrane glycoprotein, which is predicted to span the membrane seventimes with a glycosylated extracellular N-terminus and a cytoplasmicC-terminus. It is expressed in red blood cells, vascular endothelialcells and a wide range of other tissues including kidney, lung, liver,spleen, brain (Iwamoto et al., 1996, Blood, 87, 378-385) and colon(Chaudhuri et al., 1997, Blood, 89, 701-712). The Fy^(a)/Fy^(b)(FY1/FY2) blood group polymorphorism is determined by an SNP resultingin the presence of glycine (G) or aspartic acid (D), respectively, atamino acid 42 in the N-terminal extracellular domain (Iwamoto et al.,1995, Blood, 85, 622-626; Mallinson et al., 1995, British Journal ofHaematology, 90, 823-82; Tournamille et al., 1995, Human Genetics, 95,407-410). Duffy blood group system antibodies can cause haemolytictransfusion reactions (Boyland et al., 1982, Transfusion, 22, 402;Sosler et al., 1989, Transfusion, 29, 505-507) and HDFN (Vescio et al.,1987, Transfusion, 27, 366; Goodrick et al., 1997, Transfusion Medicine,7, 301-304).

The Lutheran (Lu, B-CAM, CD239) blood group antigens are carried by twosingle-pass type I (cytoplasmic C-terminus) membrane glycoproteins,which differ in the length of their cytoplasmic domains [the B-CAMglycoprotein has a shorter C-terminal cytoplasmic tail than Lu (Campbellet al., 1994, Cancer Research, 54, 5761-5765)]. The Lu glycoprotein hasfive extracellular immunoglobulin-like domains and is a member of theimmunoglobulin gene superfamily (IgSF) (Parsons et al., 1995,Proceedings of the National Academy of Science of the United States ofAmerica, 92, 5496-5500) and is expressed in red blood cells and a widerange of other tissues (Reid & Lomas-Francis, 2004, The Blood GroupAntigens Factsbook (2nd edn). Academic Press, London). The Lu^(a)/Lu^(b)(LU1/LU2) blood group antigen polymorphism is determined by a SNPresulting in the presence of histidine (H) or arginine (R),respectively, at amino acid 77 of the first predicted N-terminal IgSFdomain (El Nemer et al., 1997). Lutheran blood group system antibodieshave been reported to be involved in mild delayed haemolytic transfusionreactions (Daniels, 2002, Human Blood Groups (2nd edn). Blackwell,Oxford) but are rarely involved in HDFN (Inderbitzen et al., 1982,Transfusion, 22, 542).

Antibodies

As used herein, the term “antibody” refers to any immunoglobulin orintact molecule as well as to fragments thereof that bind to a specificepitope. Such antibodies include, but are not limited to polyclonal,monoclonal, chimeric, humanized, single chain, Fab, Fab′, F(ab)′fragments and/or F(v) portions of the whole antibody and variantsthereof. All isotypes are encompassed by this term, including IgA, IgD,IgE, IgG, and IgM.

As used herein, the term “antibody fragment” refers specifically to anincomplete or isolated portion of the full sequence of the antibodywhich retains the antigen binding function of the parent antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibody fragments.

An intact “antibody” comprises at least two heavy (H) chains and twolight (L) chains inter-connected by disulfide bonds. Each heavy chain iscomprised of a heavy chain variable region (abbreviated herein as HCVRor V_(H)) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, CH₁, CH₂ and CH₃. Each light chainis comprised of a light chain variable region (abbreviated herein asLCVR or V_(L)) and a light chain constant region. The light chainconstant region is comprised of one domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxyl-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies can mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system. The term antibody includesantigen-binding portions of an intact antibody that retain capacity tobind. Examples of binding include (i) a Fab fragment, a monovalentfragment consisting of the V_(L), V_(H), C_(L) and CH1 domains; (ii) aF(ab)₂ fragment, a bivalent fragment comprising two Fab fragments linkedby a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe V_(L) and V_(H) domains of a single arm of an antibody, (v) a dAbfragment (Ward et al., Nature, 341:544-546 (1989)), which consists of aV_(H) domain; and (vi) an isolated complementarity determining region(CDR).

As used herein, the term “single chain antibodies” or “single chain Fv(scFv)” refers to an antibody fusion molecule of the two domains of theFv fragment, V_(L) and V_(H). Although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv); see, e.g., Bird et al., Science, 242:423-426 (1988); and Hustonet al., Proc Natl Acad Sci USA, 85:5879-5883 (1988)). Such single chainantibodies are included by reference to the term “antibody” fragmentsand can be prepared by recombinant techniques or enzymatic or chemicalcleavage of intact antibodies.

As used herein, the term “human sequence antibody” includes antibodieshaving variable and constant regions (if present) derived from humangermline immunoglobulin sequences. The human sequence antibodies of theinvention can include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo). Suchantibodies can be generated in non-human transgenic animals, e.g., asdescribed in PCT App. Pub. Nos. WO 01/14424 and WO 00/37504. However,the term “human sequence antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences (e.g., humanized antibodies).

Also, recombinant immunoglobulins can be produced. See, Cabilly, U.S.Pat. No. 4,816,567, incorporated herein by reference in its entirety andfor all purposes; and Queen et al., Proc Natl Acad Sci USA,86:10029-10033 (1989).

As used herein, the term “monoclonal antibody” refers to a preparationof antibody molecules of single molecular composition. A monoclonalantibody composition displays a single binding specificity and affinityfor a particular epitope. Accordingly, the term “human monoclonalantibody” refers to antibodies displaying a single binding specificitywhich have variable and constant regions (if present) derived from humangermline immunoglobulin sequences. In one aspect, the human monoclonalantibodies are produced by a hybridoma which includes a B cell obtainedfrom a transgenic non-human animal, e.g., a transgenic mouse, having agenome comprising a human heavy chain transgene and a light chaintransgene fused to an immortalized cell.

As used herein, the term “antigen” refers to a substance that promptsthe generation of antibodies and can cause an immune response. It can beused interchangeably in the present disclosure with the term“immunogen”. In the strict sense, immunogens are those substances thatelicit a response from the immune system, whereas antigens are definedas substances that bind to specific antibodies. An antigen or fragmentthereof can be a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein can inducethe production of antibodies (i.e., elicit the immune response), whichbind specifically to the antigen (given regions or three-dimensionalstructures on the protein).

An “epitope” refers to the portion of the antigen bound by an antibody.Antigens may comprise multiple epitopes. Where the antigen is a protein,linear epitopes may range from about 5 to 20 amino acids in length.Antibodies may also recognize conformational determinants formed bynon-contiguous residues on an antigen, and an epitope can thereforerequire a larger fragment of the antigen to be present for binding, e.g.a protein domain, or substantially all of a protein sequence. It willtherefore be appreciated that a protein, which may be several hundredamino acids in length, can comprise a number of distinct epitopes.

As used herein, the term “humanized antibody,” refers to at least oneantibody molecule in which the amino acid sequence in the non-antigenbinding regions and/or the antigen-binding regions has been altered sothat the antibody more closely resembles a human antibody, and stillretains its original binding ability.

In addition, techniques developed for the production of “chimericantibodies” (Morrison, et al., Proc Natl Acad Sci, 81:6851-6855 (1984),incorporated herein by reference in their entirety) by splicing thegenes from a mouse antibody molecule of appropriate antigen specificitytogether with genes from a human antibody molecule of appropriatebiological activity can be used. For example, the genes from a mouseantibody molecule specific for an autoinducer can be spliced togetherwith genes from a human antibody molecule of appropriate biologicalactivity. A chimeric antibody is a molecule in which different portionsare derived from different animal species, such as those having avariable region derived from a murine mAb and a human immunoglobulinconstant region.

In addition, techniques have been developed for the production ofhumanized antibodies (see, e.g., U.S. Pat. No. 5,585,089 and U.S. Pat.No. 5,225,539, which are incorporated herein by reference in theirentirety). An immunoglobulin light or heavy chain variable regionconsists of a “framework” region interrupted by three hypervariableregions, referred to as complementarity determining regions (CDRs).Briefly, humanized antibodies are antibody molecules from non-humanspecies having one or more CDRs from the non-human species and aframework region from a human immunoglobulin molecule.

Alternatively, techniques described for the production of single chainantibodies can be adapted to produce single chain antibodies against animmunogenic conjugate of the present disclosure. Single chain antibodiesare formed by linking the heavy and light chain fragments of the Fvregion via an amino acid bridge, resulting in a single chainpolypeptide. Fab and F(ab′)2 portions of antibody molecules can beprepared by the proteolytic reaction of papain and pepsin, respectively,on substantially intact antibody molecules by methods that arewell-known. See e.g., U.S. Pat. No. 4,342,566. Fab′ antibody moleculeportions are also well-known and are produced from F(ab′)2 portionsfollowed by reduction of the disulfide bonds linking the two heavy chainportions as with mercaptoethanol, and followed by alkylation of theresulting protein mercaptan with a reagent such as iodoacetamide.

Antibody Assays

A number of screening assays are known in the art for assayingantibodies of interest to confirm their specificity and affinity and todetermine whether those antibodies cross-react with other proteins.

The terms “specific binding” or “specifically binding” refer to theinteraction between the antigen and their corresponding antibodies. Theinteraction is dependent upon the presence of a particular structure ofthe protein recognized by the binding molecule (i.e., the antigen orepitope). In order for binding to be specific, it should involveantibody binding of the epitope(s) of interest and not backgroundantigens.

Once antibodies are produced, they are assayed to confirm that they arespecific for the antigen of interest and to determine whether theyexhibit any cross reactivity with other antigens. One method ofconducting such assays is a sera screen assay as described in U.S. App.Pub. No. 2004/0126829, the contents of which are hereby expresslyincorporated herein by reference. However, other methods of assaying forquality control are within the skill of a person of ordinary skill inthe art and therefore are also within the scope of the presentdisclosure.

Antibodies, or antigen-binding fragments, variants or derivativesthereof of the present disclosure can also be described or specified interms of their binding affinity to an antigen. The affinity of anantibody for an antigen can be determined experimentally using anysuitable method. (See, e.g., Berzofsky et al., “Antibody-AntigenInteractions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press:New York, N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman andCompany: New York, N.Y. (1992); and methods described herein). Themeasured affinity of a particular antibody-antigen interaction can varyif measured under different conditions (e.g., salt concentration, pH).Thus, measurements of affinity and other antigen-binding parameters(e.g., K_(D), K_(a), K_(d)) are preferably made with standardizedsolutions of antibody and antigen, and a standardized buffer.

The affinity binding constant (K_(aff)) can be determined using thefollowing formula:

$K_{aff} = \frac{\left( {n - 1} \right)}{2\left( {{n\left\lbrack {mAb}^{\prime} \right\rbrack}_{t} - \lbrack{mAb}\rbrack_{t}} \right)}$

in which:

$n = \frac{\lbrack{mAg}\rbrack_{t}}{\left\lbrack {mAg}^{\prime} \right\rbrack_{t}}$

[mAb] is the concentration of free antigen sites, and [mAg] is theconcentration of free monoclonal binding sites as determined at twodifferent antigen concentrations (i.e., [mAg]_(t) and [mAg′]_(t))(Beatty et al., J Imm Meth, 100:173-179 (1987)).

The term “high affinity” for an antibody refers to an equilibriumassociation constant (K_(aff)) of at least about 1×10⁷ liters/mole, orat least about 1×10⁸ liters/mole, or at least about 1×10⁹ liters/mole,or at least about 1×10¹⁰ liters/mole, or at least about 1×10¹¹liters/mole, or at least about 1×10¹² liters/mole, or at least about1×10¹³ liters/mole, or at least about 1×10¹⁴ liters/mole or greater.“High affinity” binding can vary for antibody isotypes. K_(D), theequilibrium dissociation constant, is a term that is also used todescribe antibody affinity and is the inverse of K_(aff).

K_(D), the equilibrium dissociation constant, is a term that is alsoused to describe antibody affinity and is the inverse of K_(aff). IfK_(D) is used, the term “high affinity” for an antibody refers to anequilibrium dissociation constant (K_(D)) of less than about 1×10⁻⁷mole/liters, or less than about 1×10⁻⁸ mole/liters, or less than about1×10⁻⁹ mole/liters, or less than about 1×10⁻¹⁰ mole/liters, or less thanabout 1×10⁻¹¹ mole/liters, or less than about 1×10⁻¹² mole/liters, orless than about 1×10⁻¹³ mole/liters, or less than about 1×10⁻¹⁴mole/liters or lower.

The immunoglobulin molecules of the present invention can be of any type(e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2), or subclass of immunoglobulin molecule. In someembodiments, the antibodies are antigen-binding antibody fragments(e.g., human) and include, but are not limited to, Fab, Fab′ andF(ab′)₂, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv) and fragments comprising either a V_(L) orV_(H) domain. Antigen-binding antibody fragments, including single-chainantibodies, can comprise the variable region(s) alone or in combinationwith the entirety or a portion of the following: hinge region, CH1, CH2,and CH3 domains. Also included in the present disclosure areantigen-binding fragments comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains.

Pharmaceutical Compositions

The presently disclosed subject matter provides pharmaceuticalcompositions comprising the antibodies produced in accordance with thepresent disclosure. In some embodiments, a pharmaceutical compositioncan comprise one or more monoclonal antibodies produced using themethods disclosed herein. In some embodiments, a panel of monoclonalantibodies produced according to the present disclosure can be includedin a pharmaceutical composition.

In some embodiments a pharmaceutical composition can also contain apharmaceutically acceptable carrier or adjuvant for administration ofthe antibody. In some embodiments, the carrier is pharmaceuticallyacceptable for use in humans. The carrier or adjuvant should not itselfinduce the production of antibodies harmful to the individual receivingthe composition and should not be toxic. Suitable carriers can be large,slowly metabolized macromolecules such as proteins, polypeptides,liposomes, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers and inactive virusparticles.

Pharmaceutically acceptable salts can be used, for example mineral acidsalts, such as hydrochlorides, hydrobromides, phosphates and sulphates,or salts of organic acids, such as acetates, propionates, malonate andbenzoates.

Pharmaceutically acceptable carriers in therapeutic compositions canadditionally contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents or pH buffering substances, can be present in suchcompositions. Such carriers enable the pharmaceutical compositions to beformulated as tablets, pills, dragees, capsules, liquids, gels, syrups,slurries and suspensions, for ingestion by the patient.

The compositions of the presently disclosed subject matter can furthercomprise a carrier to facilitate composition preparation andadministration. Any suitable delivery vehicle or carrier can be used,including but not limited to a microcapsule, for example a microsphereor a nanosphere (Manome et al. (1994) Cancer Res 54:5408-5413; Saltzman& Fung (1997) Adv Drug Deliv Rev 26:209-230), a glycosaminoglycan (U.S.Pat. No. 6,106,866), a fatty acid (U.S. Pat. No. 5,994,392), a fattyemulsion (U.S. Pat. No. 5,651,991), a lipid or lipid derivative (U.S.Pat. No. 5,786,387), collagen (U.S. Pat. No. 5,922,356), apolysaccharide or derivative thereof (U.S. Pat. No. 5,688,931), ananosuspension (U.S. Pat. No. 5,858,410), a polymeric micelle orconjugate (Goldman et al. (1997) Cancer Res 57:1447-1451 and U.S. Pat.Nos. 4,551,482, 5,714,166, 5,510,103, 5,490,840, and 5,855,900), and apolysome (U.S. Pat. No. 5,922,545).

Antibody sequences can be coupled to active agents or carriers usingmethods known in the art, including but not limited to carbodiimideconjugation, esterification, sodium periodate oxidation followed byreductive alkylation, and glutaraldehyde crosslinking (Goldman et al.(1997) Cancer Res. 57:1447-1451; Cheng (1996) Hum. Gene Ther. 7:275-282;Neri et al. (1997) Nat. Biotechnol. 15:1271-1275; Nabel (1997) Vectorsfor Gene Therapy. In Current Protocols in Human Genetics, John Wiley &Sons, New York; Park et al. (1997) Adv. Pharmacol. 40:399-435;Pasqualini et al. (1997) Nat. Biotechnol. 15:542-546; Bauminger &Wilchek (1980) Meth. Enzymol. 70:151-159; U.S. Pat. No. 6,071,890; andEuropean Patent No. 0 439 095).

A therapeutic composition of the present invention comprises in someembodiments a pharmaceutical composition that includes apharmaceutically acceptable carrier. Suitable formulations includeaqueous and non-aqueous sterile injection solutions which can containanti-oxidants, buffers, bacteriostats, bactericidal antibiotics andsolutes which render the formulation isotonic with the bodily fluids ofthe intended recipient; and aqueous and non-aqueous sterile suspensionswhich can include suspending agents and thickening agents. Theformulations can be presented in unit-dose or multi-dose containers, forexample sealed ampoules and vials, and can be stored in a frozen orfreeze-dried (lyophilized) condition requiring only the addition ofsterile liquid carrier, for example water for injections, immediatelyprior to use. Some exemplary ingredients are SDS in the range of in someembodiments 0.1 to 10 mg/ml, in some embodiments about 2.0 mg/ml; and/ormannitol or another sugar in the range of in some embodiments 10 to 100mg/ml, in some embodiments about 30 mg/ml; and/or phosphate-bufferedsaline (PBS). Any other agents conventional in the art having regard tothe type of formulation in question can be used. In some embodiments,the carrier is pharmaceutically acceptable. In some embodiments thecarrier is pharmaceutically acceptable for use in humans.

Pharmaceutical compositions of the present disclosure can have a pHbetween 5.5 and 8.5, preferably between 6 and 8, and more preferablyabout 7. The pH can be maintained by the use of a buffer. Thecomposition can be sterile and/or pyrogen free. The composition can beisotonic with respect to humans. Pharmaceutical compositions of thepresently disclosed subject matter can be supplied inhermetically-sealed containers.

Pharmaceutical compositions can include an effective amount of one ormore antibodies as described herein. In some embodiments, apharmaceutical composition can comprise an amount that is sufficient totreat, ameliorate, or prevent a desired disease or condition, or toexhibit a detectable therapeutic effect. Therapeutic effects alsoinclude reduction in physical symptoms. The precise effective amount forany particular subject will depend upon their size and health, thenature and extent of the condition, and therapeutics or combination oftherapeutics selected for administration. The effective amount for agiven situation is determined by routine experimentation as practiced byone of ordinary skill in the art.

Treatment Regimens: Pharmacokinetics

The pharmaceutical compositions of the invention can be administered ina variety of unit dosage forms depending upon the method ofadministration. Dosages for typical antibody pharmaceutical compositionsare well known to those of skill in the art. Such dosages are typicallyadvisory in nature and are adjusted depending on the particulartherapeutic context or patient tolerance. The amount antibody adequateto accomplish this is defined as a “therapeutically effective dose.” Thedosage schedule and amounts effective for this use, i.e., the “dosingregimen,” will depend upon a variety of factors, including the stage ofthe disease or condition, the severity of the disease or condition, thegeneral state of the patient's health, the patient's physical status,age, pharmaceutical formulation and concentration of active agent, andthe like. In calculating the dosage regimen for a patient, the mode ofadministration also is taken into consideration. The dosage regimen mustalso take into consideration the pharmacokinetics, i.e., thepharmaceutical composition's rate of absorption, bioavailability,metabolism, clearance, and the like. See, e.g., the latest Remington's;Egleton, Peptides 18: 1431-1439, 1997; Langer, Science 249: 1527-1533,1990.

For purposes of the present invention, a therapeutically effectiveamount of a composition comprising an antibody, contains about 0.05 to1500 μg protein, preferably about 10 to 1000 μg protein, more preferablyabout 30 to 500 μg and most preferably about 40 to 300 μg, or anyinteger between these values. For example, antibodies of the inventioncan be administered to a subject at a dose of about 0.1 μg to about 200mg, e.g., from about 0.1 μg to about 5 μg, from about 5 μg to about 10μg, from about 10 μg to about 25 μg, from about 25 μg to about 50 μg,from about 50 μg to about 100 μg, from about 100 μg to about 500 μg,from about 500 μg to about 1 mg, from about 1 mg to about 2 mg, withoptional boosters given at, for example, 1 week, 2 weeks, 3 weeks, 4weeks, two months, three months, 6 months and/or a year later. It isunderstood that the specific dose level for any particular patientdepends upon a variety of factors including the activity of the specificantibody employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

Routes of administration include, but are not limited to, oral, topical,subcutaneous, intramuscular, intravenous, subcutaneous, intradermal,transdermal and subdermal. Depending on the route of administration, thevolume per dose is preferably about 0.001 to 10 ml, more preferablyabout 0.01 to 5 ml, and most preferably about 0.1 to 3 ml. Compositionscan be administered in a single dose treatment or in multiple dosetreatments on a schedule and over a time period appropriate to the age,weight and condition of the subject, the particular antibody formulationused, and the route of administration.

Kits

The invention provides kits comprising antibodies produced in accordancewith the present disclosure which can be used, for instance, fortherapeutic applications described above. The article of manufacturecomprises a container with a label. Suitable containers include, forexample, bottles, vials, and test tubes. The containers can be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which includes an active agent that is effective fortherapeutic applications, such as described above. The active agent inthe composition can comprise the antibody. The label on the containerindicates that the composition is used for a particular therapy ornon-therapeutic application, and can also indicate directions for eitherin vivo or in vitro use, such as those described above.

The following examples of specific aspects for carrying out the presentinvention are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way.

EXAMPLES Example 1: Generation of Monoclonal Antibodies Against KellAntigens

We generated mice expressing the human Kell glycoprotein (K variant) onRBCs. Transgenic RBCs were then transfused into wild-type mice, thusallowing cell surface expression without the introduction of additionalantigens. The recipient mice were pretreated with poly (I:C), which actsa an adjuvant to increase antibody responses to antigens on transfusedRBCs, as first described by Dr. Zimring (Transfusion 46(9): 1526-36,2006). Splenocytes from immunized mice were fused with myeloma partnersand monoclonal antibodies were isolated. We have isolated three clonesthat produce monoclonal IgG antibodies, which recognize the K form ofthe Kell glycoprotein but not the k form. These antibodies are usefultyping reagents for human RBCs by a variety of methods, including, butnot limited to, fluid phase agglutination, solid phase detection, tubegel detection, flow cytometry detection, enzyme linked immunoadsorbantassay, radioimmunoassay, and Western blot.

Shown in FIGS. 1-6 are the sequences of the antibodies obtained. Uponsequencing, it was determined that the antibodies designated PUMA 1 andPUMA 2 were the same. The shading indicates where the highly variableregions begin. The CDR regions of each heavy or light chain areunderlined.

The specificities of the antibodies are shown in FIGS. 7A, 7B, 8A, 8B,9A-9C, and 10A-10I. The specificities of the antibodies were determinedto be: PUMA1/2 (KEL1 or K), PUMA 3 (a common Kell epitope, PUMA 4 (KEL4or Kp^(b)). Flow cytometry was utilized to test antibody specificity byindirect immunofluorescence, using the monocolonal antibodies as theprimary reagent and a goat-anti-mouse antibody (conjugated toallophycocyanin) as a secondary antibody. Different target cellsexpressing different Kell variants were used to determine specificity.Targets included RBCs that phenotyped as homozygous for the 3 mainantithetical antigens in the Kell system, K/K, k/k, Kp^(b)/Kp^(b),Kp^(a)/Kp^(a), Js^(b)/Js^(b), Js^(a)/Js^(a). Differential binding tosuch tarets tests specificity. In the case of PUMA ½, binding was onlyobserved when K was present but not on k/k RBCs. In the case of PUMA 3,binding was observed on all RBCs regardless of phenotype for K/k,Kp^(a)/Kp^(b), or Js^(a)/Js^(b), thus indicating a common epitopeoutside these systems. However, PUMA 3 bound to only KELL glycoproteintransgenic murine RBCs and not wild-type murine RBCs; thus, the epitoperecognized by PUMA 3 is on the KELL molecule, but not K/k,Kp^(a)/Kp^(b), or Js^(a)/Js^(b). For PUMA 4, binding was only observedwhen Kp^(b) was present but not on Kp^(a)/Kp^(a) RBCs.

Example 2: Isolation of Protective Antibodies

In order to engineer a protective antibody of as disclosed herein, onemust first isolate the sequence of antibody of the correct specificityto develop the desired therapeutic. As described in Example 1, toisolate antibodies with high affinity and specificity for a given bloodgroup antigen, transgenic mice were created that express human Kellglycoprotein as a transgene; the transgenic RBCs were used as animmunogen in wild-type recipient mice. The variety of human Kellglycoprotein used expressed the KEL1, Kp^(b), and Js^(b) variants of theKEL1/KEL2, Kp^(a)/Kp^(b), and Js^(a)/Js^(b) antithetical antigens,respectively. After a high titer was achieved, spleens were harvestedfrom recipient mice, fusions were performed with a murine myeloma B cellline, and monoclonal antibodies were isolated. An anti-Kell antibodyspecific for the KEL1 variant of the KEL1/KEL2 antithetical pair wasisolated, and named PUMA1, as described in Example 1. PUMA1 was furthercharacterized as being of the IgG2a subtype and expressing a kappa lightchain variant. PUMA1 specifically recognizes the KEL1 antigen withlimited or no binding of the KEL2 variant (FIGS. 7A, 7B).

The ability of PUMA1 to induce a HTR was tested by passive immunizationof wild-type mice with PUMA1 (by intravenous tail-vein injection),followed by transfusion with murine KEL1+RBCs from KEL1 transgeneicmouse donors. Compared to a control group that got only PBS, PUMA1caused a brisk clearance of KEL1+RBCs, after which, the surviving RBCscontinued to circulate, as is typical of HTRs in both mice and also inhumans (FIG. 13). Thus, PUMA1 specifically binds to KEL1 RBCs, in vivo,with sufficient activity to induce an HTR.

Example 3: Antibody Modification

To allow engineering and manipulation of PUMA1, rapid amplification ofcDNA ends (RACE) was performed on both the heavy and light chains ofPUMA1, and the sequence for the PUMA1 antibody was elucidated (see FIGS.1 and 2). Based upon the predicted sequence, mass spectrometry wasperformed on purified monoclonal PUMA1 and predicted peptides wereconfirmed for both the heavy and light chain, demonstrating that thecorrect cDNA was amplified. The identified sequence of PUMA1 heavy chainwas cloned in frame with cDNA coding sequence for the mouse IgG3subtypes, in a eukaryotic expression vector. Similarly, the sequence ofthe PUMA1 light chain was cloned into a Eukaryotic expression vector.IgG3 was chosen, since it is typically known to have a diminishedcapacity to induce clearance of bound targets than IgG2a. The plasmidencoding PUMA1 IgG3 heavy chain was transfected into CHO cells, alongwith the expression vector for light chain, and PUMA1 IgG3 was thenpurified from culture supernatant using protein A affinitychromatography. Recombinant PUMA1 IgG2a was engineered and expressed inthe same way, to allow PUMA1 IgG2a expressed in the same system as thePUMA1 IgG3. Similar to the above murine sequences, PUMA1 has now beenhumanized by recombinant fusion of the CDRs with human IgG1, IgG2, IgG3and IgG4 (FIGS. 16A, 16B). An example of the expression of humanizedantibodies, while maintaining ability to bind RBCs is shown in FIG. 17.

Example 4: Effect of Modified Antibodies on Clearance of Transfused RedBlood Cells

To test the effects of recombinant PUMA1 IgG2a and IgG3 upon transfusedRBCs expressing the KEL1 antigen, an equivalent amount of each of thesubtypes was injected intravenously into wild-type recipient mice,followed by a transfusion with KEL1+RBCs, and the survival of theKEL1+RBCs was studied over time. Whereas recombinant IgG2a causedclearance of KEL1 RBCs (similar to the hybridoma derived PUMA1 IgG2a,recombinant PUMA1 IgG3 caused only a small amount of clearance (FIG.14). To test if the IgG3 had the ability to block hemolysis caused byIgG2a, recipient mice were infused first with the hemolytic form ofPUMA1 (IgG2a) and were then infused with the less hemolytic form (IgG3),followed by a transfusion with KEL1+ RBCs (FIG. 15—blue (left) bars).Alternatively, PUMA1 IgG3 was added to the RBC unit of KEL1+ RBCs priorto transfusion into a recipient pre-immunized with PUMA1 IgG2a (FIG.15—red (right) bars). In either case, the addition of an excess of PUMA1IgG3 decreased the clearance seen with IgG2a to the levels typicallyseen with IgG3. These data demonstrate that injection of a form ofanti-KEL1 with diminished capacity to remove KEL1+RBCs can reverse theeffects of pre-existing hemolytic PUMA1 antibodies.

Together, the data presented herein demonstrate the efficacy of using aless hemolytic form of an antibody to an RBC antigen to preventclearance of transfused RBCs by a more hemolytic form. The efficacy ofthis approach is achievable either by directly injecting the blockingantibody into the recipient, prior to transfusion with incompatibleRBCs, or by pre-incubating the RBCs with antibody before transfusing.The former approach has the theoretical advantage that pre-incubationwith RBCs is not a requirement, and thus is a more rapid treatment inurgent situations. The latter approach, of pre-incubation, may beadvantageous (when time allows) due to the ability of blocking antibodyto equilibrate with the antigen on the offending RBCs. However, thepre-incubation approach also runs the risk of inducing agglutination ofthe antigen positive RBCs (likely a variable issue on an antigen byantigen and antibody by antibody basis). While agglutination does notappear to be a risk in the current experimental system, this can beempirically tested on a range of human RBC units for any givenantigen/antibody pair to assess if it is a problem. Should agglutinationbe a problem, it can be likely remedied either by pre-injecting antibodyinto the recipient, or in extreme cases, by the engineering ofmonovalent antigen binding molecules.

Example 5: Further Engineering of HTR Blocking Antibodies

As shown above, we have isolated an anti-KEL1 monoclonal antibodysequence. We have performed recombinant manipulation of the antibody toswitch the constant region for a less hemolytic form, which results in atherapeutic that can interfere with a HTR, either by injecting into therecipient, or through pre-incubation with the transfused RBCs.Recombinant manipulation of the antibody to switch the constant regionfor a less hemolytic form results in a therapeutic that can interferewith a HTR, either by injecting into the recipient, or throughpre-incubation with the transfused RBCs.

Based on the foregoing which demonstrate efficacy of the approach, andour demonstration of successful humanization (see FIGS. 16A, 16B, and17), one may introduce additional modifications of the PUMA1 heavy andlight chains which include the following: modification of the Fc domainto eliminate effector function (e.g. removing FcgR binding activity andcomplement fixing activity); addition of in frame domains to preventand/or suppress immune responses; and addition of chemical moieties toprevent and/or suppress immune responses.

While specific aspects of the invention have been described andillustrated, such aspects should be considered illustrative of theinvention only and not as limiting the invention as construed inaccordance with the accompanying claims.

All publications and patent applications cited in this specification areherein incorporated by reference in their entirety for all purposes asif each individual publication or patent application were specificallyand individually indicated to be incorporated by reference for allpurposes.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications can be made thereto without departing from the spiritor scope of the appended claims.

What is claimed:
 1. An isolated antibody or fragment thereof that bindsto a Kell blood group antigen KEL1 (K) and blocks a hemolytictransfusion reaction, which isolated antibody or fragment thereofcomprises: a heavy chain comprising: CDR1 shown in SEQ ID NO: 25, CDR2shown in SEQ ID NO: 26, and CDR3 shown in SEQ ID NO: 27; and a lightchain comprising: CDR1 shown in SEQ ID NO: 28, CDR2 shown in SEQ ID NO:29, and CDR3 shown in SEQ ID NO:
 30. 2. The antibody or fragment ofclaim 1, wherein the antibody or fragment recognizes a single amino acidpolymorphism.
 3. The antibody or fragment thereof according to claim 1,wherein the antibody or fragment thereof is selected from the groupconsisting of: (a) a whole immunoglobulin molecule; (b) an scFv; (c) aFab fragment; (d) an F(ab′)₂; and (e) a disulfide linked Fv.
 4. Theantibody or fragment thereof according to claim 1, which comprises aheavy chain immunoglobulin constant domain selected from the groupconsisting of: (a) a human IgM constant domain; (b) a human IgG1constant domain; (c) a human IgG2 constant domain; (d) a human IgG3constant domain; (e) a human IgG4 constant domain; and (f) a humanIgA1/2 constant domain.
 5. The antibody or fragment thereof according toclaim 1, which comprises a light chain immunoglobulin constant domainselected from the group consisting of: (a) a human Ig kappa constantdomain; and (b) a human Ig lambda constant domain.
 6. The antibody orfragment thereof according to claim 1, wherein the antibody or fragmentthereof is a mouse IgG1, IgG2a, IgG2b, IgG2c, or IgG3.
 7. The antibodyor fragment thereof according to claim 1, wherein the antibody orfragment thereof binds to an antigen with an affinity constant (K_(D))of less than 1×10⁻⁸ M.
 8. The antibody or fragment thereof according toclaim 1, wherein the heavy chain comprises SEQ ID NO:
 1. 9. The antibodyor fragment thereof according to claim 1, wherein the light chaincomprises SEQ ID NO:
 5. 10. The antibody or fragment thereof accordingto claim 1, wherein the heavy chain is selected from SEQ ID NOs: 21-24.11. The antibody or fragment thereof according to claim 6, wherein theantibody or fragment thereof is a mouse IgG2a.
 12. The antibody orfragment thereof according to claim 6, wherein the antibody or fragmentthereof is a mouse IgG3.
 13. The antibody or fragment thereof accordingto claim 1, wherein the heavy chain further comprises modification of Fcdomain to eliminate effector function.
 14. The antibody or fragmentthereof according to claim 1, wherein the heavy chain and/or light chaincomprise in frame domains to suppress immune responses.
 15. The antibodyor fragment thereof according to claim 1, wherein the heavy chain and/orlight chain comprise chemical moieties to suppress immune responses.